U.S. patent application number 16/681822 was filed with the patent office on 2021-05-13 for method for treating a cancer associated with the activation of galectin-1.
This patent application is currently assigned to TRINEO BIOTECHNOLOGY CO. LTD. The applicant listed for this patent is TRINEO BIOTECHNOLOGY CO. LTD. Invention is credited to Mon-Tarng CHEN, Su-Yu CHEN, Teng-Hai CHEN, Cheng-Po HUANG, Chih-Yuan LIAO, Yi-Hsiu LIN, Chien-Yuan WANG, Ssu-Chia WANG.
Application Number | 20210137945 16/681822 |
Document ID | / |
Family ID | 1000004488426 |
Filed Date | 2021-05-13 |
United States Patent
Application |
20210137945 |
Kind Code |
A1 |
CHEN; Teng-Hai ; et
al. |
May 13, 2021 |
METHOD FOR TREATING A CANCER ASSOCIATED WITH THE ACTIVATION OF
GALECTIN-1
Abstract
Provided herein is a method for the treatment and/or prophylaxis
of a cancer associated with galectin-1. The method includes
administering to a subject a pharmaceutical composition that mainly
composed of ganoderic acid S (GAS) and ganoderic acid T (GAT). The
method further includes administering to the subject another
anti-cancer agent before, together with, or after the
administration of the present pharmaceutical composition, so as to
synergistically suppress the growth of the cancer.
Inventors: |
CHEN; Teng-Hai; (Tainan
City, TW) ; CHEN; Mon-Tarng; (Tainan City, TW)
; WANG; Chien-Yuan; (Tainan City, TW) ; HUANG;
Cheng-Po; (New Taipei City, TW) ; CHEN; Su-Yu;
(New Taipei City, TW) ; LIN; Yi-Hsiu; (New Taipei
City, TW) ; WANG; Ssu-Chia; (New Taipei City, TW)
; LIAO; Chih-Yuan; (New Taipei City, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TRINEO BIOTECHNOLOGY CO. LTD |
New Taipei City |
|
TW |
|
|
Assignee: |
TRINEO BIOTECHNOLOGY CO.
LTD
New Taipei City
TW
|
Family ID: |
1000004488426 |
Appl. No.: |
16/681822 |
Filed: |
November 13, 2019 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/337 20130101;
A61K 31/7048 20130101; A61K 31/704 20130101; A61P 35/00 20180101;
A61K 31/575 20130101 |
International
Class: |
A61K 31/575 20060101
A61K031/575; A61K 31/337 20060101 A61K031/337; A61K 31/704 20060101
A61K031/704; A61K 31/7048 20060101 A61K031/7048; A61P 35/00
20060101 A61P035/00 |
Claims
1. A method of treating a cancer associated with the activation of
galectin-1 in a subject comprising administering to the subject an
effective amount of a pharmaceutical composition consisting
essentially of ganoderic acid S (GAS) and ganoderic acid T (GAT);
and a pharmaceutically acceptable excipient.
2. The method of claim 1, wherein the GAS and the GAT are present
in the pharmaceutical composition in a weight ratio from about 1:1
to 1:10.
3. The method of claim 1, wherein the GAS and the GAT are present
in the pharmaceutical composition in a weight ratio of about
1:2.
4. The method of claim 1, wherein the cancer associated with the
activation of galectin-1 is selected from the group consisting of
glioblastoma, thyroid cancer, gastrointestinal cancer, liver
cancer, lung cancer, breast cancer, pancreatic cancer, melanoma,
prostate cancer, ovarian cancer, adenocarcinoma, bladder cancer and
kidney cancer.
5. The method of claim 4, wherein the cancer associated with the
activation of galectin-1 is ovarian cancer.
6. The method of claim 1, further comprising administrating an
anti-cancer drug to the subject.
7. The method of claim 6, wherein the anti-cancer drug is selected
from the group consisting of paclitaxel, docetaxel, camptothecin
(CPT), topotecan (TPT), irinotecan (CPT-11), doxorubicin,
daunorubicin, epirubicin, fluorouracil, cisplatin,
cyclophosphamide, vinblastine, vincristine, ifosfamide, melphalan,
mitomycin, methotrexate, mitoxantrone, teniposide, etoposide,
bleomycin, leucovorin, cytarabine, dactinomycin, streptozocin,
combretastatin A4-phosphate, and SU5416.
8. The method of claim 7, wherein the anti-cancer drug is
paclitaxel.
9. The method of claim 7, wherein the anti-cancer drug is
doxorubicin.
10. The method of claim 7, wherein the anti-cancer drug is
etoposide.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
[0001] The present disclosure relates to a method for treating or
preventing a cancer associated with activation of galectin-1 using
the pharmaceutical composition comprising a novel combination of
triterpenoids, ganoderic acid S and ganoderic acid T.
2. Description of Related Art
[0002] Galectin-1 has been implicated in cancer progression,
invasion and metastasis. Evidence indicates that galectin-1
participates in tumor progression by evoking T cell energy and
contributes to cancer-immune escape. Galectin-1 also relates to
tumor endothelial cell adhesion and migration; therefore,
Galectin-1 may act as a target for therapeutic intervention against
cancer.
[0003] Inventors of the present disclosure unexpectedly identified
certain ganoderic acids could suppress the progression and
metastasis of a cancer mediated by the activation of galectin-1,
thus these ganoderic acids may serve as candidates for the
development of a medicament for the treatment of cancers associated
with the activation of galectin-1.
SUMMARY
[0004] The present disclosure is based, at least in part,
unexpected discovery that certain triterpenoids or ganoderic acids
isolated from the fruit bodies or mycelia of Ganoderma lucidum may
suppress or inhibit the growth of cancerous cells associated with
the activation of galectin-1. Thus, these compounds may serve as
candidates for the development of a medicament for the treatment
and/or prophylaxis of cancers associated with the activation of
galectin-1.
[0005] Accordingly, it is the aspect of this disclosure to provide
a method of treating or preventing a cancer associated with the
activation of galectin-1 in a subject. The method includes the step
of administering to the subject an effective amount of a
pharmaceutical composition consisting essentially of ganoderic acid
S (GAS) and ganoderic acid T (GAT); and a pharmaceutically
acceptable excipient. According to embodiments of the present
disclosure, the GAS and the GAT are present in the pharmaceutical
composition in a weight ratio from about 1:1 to 1:10. Preferably,
the GAS and GAT are present in the pharmaceutical composition in
the weight ratio of 1:2.
[0006] The cancer associated with galectin-1 treatable by the
present method may be any of glioblastoma, thyroid cancer,
gastrointestinal cancer, liver cancer, lung cancer, breast cancer,
pancreatic cancer, melanoma, prostate cancer, ovarian cancer,
adenocarcinoma, bladder cancer and kidney cancer.
[0007] Alternatively or optionally, the method further includes the
step of administrating to the subject an anti-cancer drug before,
together with, or after the administration of the present
pharmaceutical composition, so as to synergistically suppress the
growth of the cancer.
[0008] Examples of the anti-cancer drug suitable for use in the
present method include, but are not limited to, paclitaxel,
docetaxel, camptothecin (CPT), topotecan (TPT), irinotecan
(CPT-11), doxorubicin, daunorubicin, epirubicin, fluorouracil,
cis-platin, cyclophosphamide, vinblastine, vincristine, ifosfamide,
melphalan, mitomycin, methotrexate, mitoxantrone, teniposide,
etoposide, bleomycin, leucovorin, cytarabine, dactinomycin,
streptozocin, combretastatin A4-phosphate, and SU5416. In one
preferred embodiment, synergistic suppression of the ovarian cancer
is achieved when the present pharmaceutical composition is
administered together with the anti-cancer drug of paclitaxel. In
another preferred embodiment, synergistic suppression of the
ovarian cancer is achieved when the present pharmaceutical
composition is administered together with the anti-cancer drug of
doxorubicin.
[0009] Alternatively or optionally, the method further includes the
step of subjecting the subject to another treatment that is any of
a surgical operation or radiation treatment before, concurrently
with or after the step of administering the pharmaceutical
composition of this disclosure to the subject.
[0010] The details of one or more embodiments of this disclosure
are set forth in the accompanying description below. Other features
and advantages of the invention will be apparent from the detail
descriptions, and from claims.
[0011] It is to be understood that both the foregoing general
description and the following detailed description are by examples,
and are intended to provide further explanation of the invention as
claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0013] The present description will be better understood from the
following detailed description read in light of the accompanying
drawings, where:
[0014] FIG. 1 illustrates the effects of TN-GL-01 composition of
present invention on ovarian cancer ES-2 cells in accordance with
one embodiment of present invention;
[0015] FIG. 2 illustrates the effects of TN-GL-01 composition on
the expression of galectin-1 via western blot analysis in
accordance with one embodiment of present invention;
[0016] FIG. 3 illustrates the respective effects of TN-GL-01
composition, cisplatin, and doxorubicin on paclitaxel-induced cell
death in accordance with one embodiment of present invention;
[0017] FIG. 4 illustrates the respective effects of TN-GL-01
composition, cisplatin, and doxorubicin on doxorubicin-induced cell
death in accordance with one embodiment of present invention;
[0018] FIG. 5A are photographs illustrating the results of
immunohistochemical analysis and the tumor appearances in
accordance with one embodiment of present invention;
[0019] FIG. 5B is a bar graph depicting the weight of tumors of
FIG. 5B;
[0020] FIG. 6A are photographs illustrating the results of
immunohistochemical analysis on CD8-positive cells in accordance
with one embodiment of present invention;
[0021] FIG. 6B is a bar graph depicting the percentage of human
immune cells in the cancerous tissues in accordance with one
embodiment of present invention;
[0022] FIG. 7A is a bar graph depicting the amount of paclitaxel
retained in the cancerous tissues in accordance with one embodiment
of present invention; and
[0023] FIG. 7B is a bar graph depicting the weight of tumors of
FIG. 7A.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The detailed description provided below in connection with
the appended drawings is intended as a description of the present
disclosure and is not intended to represent the only forms in which
the present disclosure may be constructed or utilized.
1. Definition
[0025] For convenience, certain terms employed in the context of
the present disclosure are collected here. Unless defined
otherwise, all technical and scientific terms used herein have the
same meaning as commonly understood by one of the ordinary skill in
the art to which this invention belongs.
[0026] The terms "treatment" and "treating" are used herein to
include preventative (e.g., prophylactic), curative, or palliative
treatment that results in a desired pharmaceutical and/or
physiological effect. Preferably, the effect is therapeutic in
terms of partially or completely curing or preventing the growth of
tumor cells. Also, the term "treating" as used herein refers to
application or administration of the pharmaceutical composition of
the present disclosure to a subject, who has a medical condition, a
symptom of the condition, a disease or disorder secondary to the
condition, or a predisposition toward the condition, with the
purpose to partially or completely alleviate, ameliorate, relieve,
delay onset of, inhibit progression of, reduce severity of, and/or
reduce incidence of one or more symptoms or features of a
particular disease, disorder, and/or condition. Treatment may be
administered to a subject who does not exhibit signs of a disease,
disorder, and/or condition and/or to a subject who exhibits only
early signs of a disease, disorder, and/or condition for the
purpose of decreasing the risk of developing pathology associated
with the disease, disorder, and/or condition. As used herein, the
symptom, disease, disorder or condition may be solid tumor or
metastatic tumor. Treatment is generally "effective" if one or more
symptoms or clinical markers are reduced as that term is defined
herein.
[0027] The term "prophylaxis" as used herein means prevention
against a future event. In the context of prophylaxis against tumor
or tumor metastasis that may potentially occur as a consequence of
a surgical or diagnostic procedure, the prophylactic administration
can occur before, contemporaneous with, and/or after the
procedure.
[0028] The term "an effective amount" as used herein refers to an
amount effective, at dosages, and for periods of time necessary, to
achieve the therapeutically desired result with respect to the
treatment of cancer.
[0029] The terms "compounds," "compositions," "agent" and
"medicament" are used interchangeably herein to refer to a compound
or a composition of which, when administered to a subject such as a
human or an animal induces a desired pharmacological and/or
physiological effect by local and/or systemic action.
[0030] The terms "administered," "administering" and
"administration" are used interchangeably herein to refer means
either directly administering a compound or a composition of the
present invention, or administering a prodrug, derivative or analog
which will form an equivalent amount of the active compound within
the body.
[0031] The term "subject" or "patient" refers to an animal
including the human species that is treatable with the compositions
and/or methods of the present invention. The term "subject" or
"patient" intended to refer to both the male and female gender
unless one gender is specifically indicated. Accordingly, the term
"subject" or "patient" includes, but is not limited to, human,
non-human primate such as any mammal, dog, cat, horse, sheep, pig,
cow, and etc., preferably a human, which may benefit from treatment
by the compound of this disclosure. The terms "subject" and
"patient" are used interchangeably in the present disclosure.
[0032] Notwithstanding that the numerical ranges and parameters
setting forth the broad scope of the invention are approximations,
the numerical values set forth in the specific examples are
reported as precisely as possible. Any numerical value, however,
inherently contains certain errors necessarily resulting from the
standard deviation found in the respective testing measurements.
Also, as used herein, the term "about" generally means within 10%,
5%, 1%, or 0.5% of a given value or range. Alternatively, the term
"about" means within an acceptable standard error of the mean when
considered by one of ordinary skill in the art. Other than in the
operating/working examples, or unless otherwise expressly
specified, all of the numerical ranges, amounts, values and
percentages such as those for quantities of materials, durations of
times, temperatures, operating conditions, ratios of amounts, and
the likes thereof disclosed herein should be understood as modified
in all instances by the term "about." Accordingly, unless indicated
to the contrary, the numerical parameters set forth in the present
disclosure and attached claims are approximations that can vary as
desired. At the very least, each numerical parameter should at
least be construed in light of the number of reported significant
digits and by applying ordinary rounding techniques.
2. The Pharmaceutical Composition
[0033] The present disclosure is based, at least in part,
unexpected discovery that a novel combination of certain ganoderic
acids isolated from the fruit bodies or mycelia of Ganoderma
lucidum, may suppress or inhibit the expression of galectin-1,
which in term, leads to the suppression of the tumor. Accordingly,
the novel combination of the identified ganoderic acids,
particularly, the combination of ganoderic acid S (GAS) and
ganoderic acid T (GAT), is useful as a therapeutic medicament for
the treatment or prophylaxis of cancers mediated by galectin-1.
More unexpectedly, a synergistic suppression of the growth and/or
metastasis of a tumor is achieved when the present combination of
GAS and GAT is administered with an anti-cancer drug, such as
etoposide, doxorubicin, and paclitaxel.
[0034] Accordingly, it is the first aspect of this disclosure to
provide a pharmaceutical composition for the treatment or
prophylaxis of a cancer associated with the activation of
galectin-1. The pharmaceutical composition includes a triterpenoid
that consists essentially of ganoderic acid S (GAS) and ganoderic
acid T (GAT); and a pharmaceutically acceptable excipient.
[0035] In general, GAS and GAT may be respectively isolated from
Ganoderma spp in accordance with any method known in the art, such
as from the fruit bodies of Ganoderma lucidum in accordance with
the method described by Hirotani et al (Phytochemistry (1987),
26(10), 2797-2803). Alternatively, GAS and GAT may be isolated from
the cultivating waste of Ganoderma lucidum, particularly from the
mycelium remained in the cultivating media of Ganoderma lucidum
after harvest, in accordance with the method described in Taiwan
Patent No. 1381844, the disclosure of which is incorporated herein
by reference. The isolation in general involves extracting the
Ganoderma spp with a solvent, preferably an alcoholic solution, at
a temperature above room temperature; followed by column
chromatography purification, which may be high performance liquid
chromatography (HPLC), reverse phase liquid chromatography and
etc.; concentrating and drying the isolated product, until a dried
powder is produced.
[0036] According to embodiments of the present disclosure, the GAS
and the GAT are respectively present in the pharmaceutical
composition in a ratio from about 1:1 to 1:10 by weight, such as
1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10 by weight. In
one preferred embodiment, the GAS and the GAT exist in the
pharmaceutical composition in a ratio of about 1:2 by weight.
[0037] The triterpenoid of the present disclosure, i.e., the
combination of GAS and GAT, is present at a level of about 0.1% to
99% by weight, based on the total weight of the pharmaceutical
composition, such as about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,
0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,
53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,
70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% by weight in
the pharmaceutical composition. In some embodiments, the
triterpenoid of the present disclosure is present at a level of at
least 1% by weight, based on the total weight of the pharmaceutical
composition. In certain embodiments, the triterpenoid of the
present disclosure is present at a level of at least 5% by weight,
based on the total weight of the pharmaceutical composition. In
still other embodiments, the triterpenoid of the present disclosure
is present at a level of at least 10% by weight, based on the total
weight of the pharmaceutical composition. In still yet other
embodiments, the triterpenoid is present at a level of at least 25%
by weight, based on the total weight of the pharmaceutical
composition.
[0038] In some embodiments, the medicament or the pharmaceutical
composition of the present disclosure is used as an adjuvant
therapy, in addition to the major cancer therapy, which includes,
but is not limited to, surgical operation, radiotherapy, or
chemotherapy. In some embodiments, the present pharmaceutical
composition is used together with a chemotherapeutic agent or an
anti-cancer agent.
[0039] The medicament or said pharmaceutical composition is
prepared in accordance with acceptable pharmaceutical procedures,
such as described in Remington's Pharmaceutical Sciences, 17.sup.th
edition, ed. Alfonoso R. Gennaro, Mack Publishing Company, Easton,
Pa. (1985). Pharmaceutically acceptable excipients are those that
are compatible with other ingredients in the formulation and
biologically acceptable.
[0040] The pharmaceutical composition of the present disclosure may
be administered by any suitable route, for example, orally in
capsules, suspensions or tablets or by parenteral administration.
Parenteral administration can include, for example, systemic
administration such as intramuscular, intravenous, subcutaneous, or
intraperitoneal injection. In preferred embodiments, the
pharmaceutical composition of the present disclosure are
administered orally (e.g., dietary) to the subject.
[0041] For oral administration, the pharmaceutical composition of
the present disclosure may be formulated into tablets containing
various excipients such as microcrystalline cellulose, sodium
citrate, calcium carbonate, dicalcium phosphate, and glycine; along
with various disintegrants such as starch, alginic acid and certain
silicates; together with granulation binders like
polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally,
lubricating agents such as magnesium stearate, sodium lauryl
sulfate and talc may be added. Solid composition may also be
employed as fillers in gelatin capsules; preferred materials in
this connection also include lactose or milk sugar as well as high
molecular weight polyethylene glycols. When aqueous suspensions
and/or elixirs are desired for oral administration, the active
ingredient may be combined with various sweetening or flavoring
agents, coloring matter or dyes, and if so desired, emulsifying
and/or suspending agents as well, together with diluents such as
water, ethanol, propylene glycol, glycerin and a combination
thereof.
[0042] For parenteral administration, the medicament or
pharmaceutical composition of the present disclosure may be
formulated into liquid pharmaceutical compositions, which are
sterile solutions, or suspensions that can be administered by, for
example, intravenous, intramuscular, subcutaneous, or
intraperitoneal injection. Suitable diluents or solvent for
manufacturing sterile injectable solution or suspension include,
but are not limited to, 1,3-butanediol, mannitol, water, Ringer's
solution, and isotonic sodium chloride solution. Fatty acids, such
as oleic acid and its glyceride derivatives are also useful for
preparing injectables, as are natural pharmaceutically acceptable
oils, such as olive oil or castor oil. These oil solutions or
suspensions may also contain alcohol diluent or carboxymethyl
cellulose or similar dispersing agents. Other commonly used
surfactants such as Tweens or Spans or other similar emulsifying
agents or bioavailability enhancers that are commonly used in
manufacturing pharmaceutically acceptable dosage forms can also be
used for the purpose of formulation.
[0043] It will be appreciated that the dosage of the pharmaceutical
composition of the present disclosure will vary from patient to
patient not only for the particular route of administration, and
the ability of the composition to elicit a desired response in the
patient, but also factors such as disease state or severity of the
condition to be alleviated, age, sex, weight of the patient, the
state of being of the patient, and the severity of the pathological
condition being treated, concurrent medication or special diets
then being followed by the patient, and other factors which those
skilled in the art will recognize, with the appropriate dosage
ultimately being at the discretion of the attendant physician.
Dosage regimens may be adjusted to provide the improved therapeutic
response. An effective amount is also one in which any toxic or
detrimental effects of the composition are outweighed by the
beneficial effects. Preferably, the compositions of the present
disclosure are administered at a dosage and for a time such that
the number and/or severity of the symptoms are decreased.
3. The Treatment Method
[0044] The present disclosure also provides a method of treating a
cancer associated with the activation of galectin-1 in a subject.
The method includes the step of administering to the subject an
effective amount of the pharmaceutical composition described
above.
[0045] Alternatively or optionally, the method further includes the
step of administering another agent known to improve the treatment
of cancer, before, together with and/or after administering the
pharmaceutical composition of this invention. Examples of such
agent include, but are not limited to, anti-cancer drug,
anti-angiogenesis agent, anti-virus agent, analgesic, anti-anemia
drug, cytokine, granulocyte colony-stimulating factor (G-CSF), and
anti-nausea drug and the like.
[0046] In some preferred embodiments, the method further includes
administering to the subject an anti-cancer drug in addition to the
administration of the present pharmaceutical composition, in which
a synergistic effect on the suppression of cancers is achieved via
suppressing the expression of galectin-1. Examples of anti-cancer
drug suitable for use in the present method include, but are not
limited to, paclitaxel, docetaxel, camptothecin (CPT), topotecan
(TPT), irinotecan (CPT-11), Doxorubicin, daunorubicin, epirubicin,
fluorouracil, cis-platin, cyclophosphamide, vinblastine,
vincristine, ifosfamide, melphalan, mitomycin, methotrexate,
mitoxantrone, teniposide, etoposide, bleomycin, leucovorin,
cytarabine, dactinomycin, streptozocin, combretastatin
A4-phosphate, SU5416, and the like. Examples of anti-angiogenesis
agent include, but are not limited to, DS 4152, TNP-470, SU6668,
endostatin, 2-methoxyestradiol, angiostatin, thalidomide,
tetrathiomolybdate, linomide, IL-12, and the like. Examples of
anti-virus agent include, but are not limited to, amantadine,
rimantadine, and the like. Examples of analgesic include, but are
not limited to, paracetamol such as para-acetylaminophenol,
non-steroidal anti-inflammatory drug (NSAID) such as salicylates,
and opioid drugs such as morphine and opium. Example of anti-anemia
drug includes, but is not limited to, erythropoietin
[0047] Examples of cancers treatable by the present method include,
but are not limited to, glioblastoma, thyroid cancer,
gastrointestinal cancer, liver cancer, lung cancer, breast cancer,
pancreatic cancer, melanoma, prostate cancer, ovarian cancer,
adenocarcinoma, bladder cancer and kidney cancer.
[0048] In one preferred embodiment, the pharmaceutical composition
is administered together with paclitaxel to a subject suffering
from ovarian cancer. In another preferred the pharmaceutical
composition is administered together with doxorubicin to a subject
suffering from ovarian cancer.
[0049] In the present method, the pharmaceutical composition
described above is administered to the subject in an amount of
about 1 to 120 mg/Kg body weight of the subject per day, such as 1,
10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120 mg/Kg/day;
preferably, in the amount of about 30 to 110 mg/Kg/day, such as 30,
40, 50, 60, 70, 80, 90, 100 or 110 mg/Kg/day; more preferably, in
the amount of 80 to 100 mg/Kg/day, such as 80, 90, or 100
mg/Kg/day. It will be appreciated that the dosage of the
pharmaceutical composition of the present disclosure will vary from
patient to patient not only for the particular route of
administration, and the ability of the composition to elicit a
desired response in the patient, but also factors such as disease
state or severity of the condition to be alleviated, age, sex,
weight of the patient, the state of being of the patient, and the
severity of the pathological condition being treated, concurrent
medication or special diets then being followed by the patient, and
other factors which those skilled in the art will recognize, with
the appropriate dosage ultimately being at the discretion of the
attendant physician. Dosage regimens may be adjusted to provide the
improved therapeutic response. An effective amount is also one in
which any toxic or detrimental effects of the composition are
outweighed by the therapeutically beneficial effects. The dose can
be administered in a single dosage, or alternatively in more than
one dosage, such as 2, 3, or 4 dosages a day.
[0050] In some embodiments, the method further includes subjecting
the subject to a radiation treatment after the administration of
the pharmaceutical composition of present invention.
[0051] The present invention will now be described more
specifically with reference to the following embodiments, which are
provided for the purpose of demonstration rather than
limitation.
Examples
Materials and Methods
[0052] Cell Culture
[0053] Ovarian cancer cell ES-2 and myeloma cell line ARH-77 were
used in the present disclosure. Cells was cultured and maintained
in Dulbecco's modified Eagle media (DMEM) supplemented with 10%
fetal bovine serum (FBS), 100 IU/ml penicillin, 100 ng/ml
streptomycin, 2 mM glutamine, non-essential amino acids and sodium
pyruvate in 5% CO.sub.2 at 37.degree. C. Cells were grown and
maintained in Petri dishes (each was 10 cm in diameter) until they
reached 80% confluence, and then were subject to cell passages.
Briefly, cells were first washed with phosphate buffer solution
(PBS, 3 ml) once, then treated with 0.05% Trypsin/0.025% EDTA
solution (1 mL) for 5 min so that the attached cells become
suspended. The suspended cells were harvested and 2 mL fresh
culture media were added therein to neutralize any remaining
activity of trypsin. Cell density was adjusted by adding
appropriate amounts of culture medium to the cell suspension, which
was then used to seed the culture plates. The plates were then
returned to the incubator, and cultured in accordance with the
steps described above.
[0054] Cell Activity Analysis
[0055] Cells were seeded in 96-well plates with a density of 3,000
cells/well and cultured in accordance with the procedures described
above. On the day when cell activity analysis was to be conducted,
cells were first treated with various concentrations of the
triterpenoids of the present disclosure, imatinib or gefitinib for
at least 48 hrs, before subjecting them to MTT assay.
[0056] MTT Assay
[0057] 50 .mu.L of the MTT stock solution (5 mg MTT dissolved in 1
ml of sterile PBS) was added to each well, and 50 .mu.L of the MTT
stock solution was added to 500 .mu.L of medium alone and used as a
negative control. Each sample was incubated at 37.degree. C. for 4
hours. Aliquots (450 .mu.l) from each sample were taken to a new
well of 48-well culture plate, adding 1004, DMSO, mixing thoroughly
using the pipette and reacted at 37.degree. C. for 20 min and
absorbance was measured at 570 nm.
[0058] Immunohistochemical Analysis
[0059] Tissues were fixed by 4% paraformaldehyde, embedded in
paraffin. Tissue sections of ES2 tumor were processed for antigen
retrieval. The samples were incubated using Ku80 (C48E7) rabbit
monoclonal antibody, following by incubation with the anti-rabbit
HRP secondary antibody. Samples were stained using DAB (brown) and
counterstained with hematoxylin (blue).
[0060] Western Blot Analysis
[0061] Samples were respectively subjected to SDS-PAGE, transferred
to PVDF membranes. These blots were blocked with 5% nonfat milk
powder in TBS-0.1% Tween-20 for 30 minutes, followed by incubation
with primary antibodies at 4.degree. C. overnight and then
horseradish peroxidase-conjugated secondary antibodies (Amersham
Biosciences, Piscataway). Imaging of bands was performed using
Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific)
and ImageQuant LAS-4000 (GE Healthcare).
[0062] Animal Model of Primary Ovarium Tumor
[0063] Male NOD/SCID mice about 6 weeks old were used in this
experiment. Animals were kept at specific pathogen-free conditions
under 12:12 light-dark cycle with food (laboratory rodent diet 5001
purchased from PMI Nutrition International Inc. MO, USA) and water
(i.e., distilled water) provided ad libitum, ambient temperature
and relative humidity were respectively set at 22.+-.3.degree. C.
and 50.+-.20%. All procedures involving animal studies of the
present disclosure comply with the "Guideline for the Care and Use
of Laboratory Animals" issued by The Chinese-Taipei Society of
Laboratory Animal Sciences.
[0064] The day before tumor inoculation, mice were randomly divided
into groups while making sure that the average weight in each group
did not differ significantly. The fur on the right hind leg of each
animal was then shaved. To inoculate tumor in mice, ovarian cancer
cells ES-2 (1.times.10.sup.7 cancer cells/0.1 mL) were injected
subcutaneously to NOD/SCID mice, to generate subcutaneous (s.c.)
xenograft tumors.
Example 1 Preparation and Characterization of the Present
Pharmaceutical Composition
[0065] 1.1 Preparation of the Present Pharmaceutical
Composition
[0066] A pharmaceutical composition, herein "TN-GL-01 composition"
was prepared by mixing the dried powders of GAT (26 mg), GAS (14
mg) and crystallized methylcellulose (MC) (60 mg) in a blender
until a homogeneous mixture was obtained.
[0067] 1.2 TN-GL-01 Composition Inhibited the Activity of Orarian
Cancer Cells
[0068] The effects of the TN-GL-01 composition of example 1.1 on
ovarian cancer cells (i.e., ES-2 cells) were evaluated by MTT assay
in accordance with procedures described in the "Material and
Methods" section. Results are provided in FIG. 1.
[0069] As depicted, 50% inhibition of ES-2 cells by the TN-GL-01
composition, GAS and GAT (IC.sub.50) were independently 30.1, 58.3
and 24.5 .mu.g/mL. Thus, as expected, TN-GL-01 composition of
example 1.1, GAS, and GAT all exhibited cytotoxic effect on ES-2
cells.
[0070] 1.3 TN-GL-01 Composition Inhibited Galectin-1 Expression of
ES-2 Cells
[0071] In this example, the effects of TN-GL-01 composition on the
expression of galectin-1 in ERK (extracellular-signal-regulated
kinase) pathway was investigated via western blot analysis. Results
are shown in FIG. 2. As depicted, GAS, GAT, and TN-GL-01
composition were all effective in suppressing the expression of
galectin-1. Thus, it was concluded that the present TN-GL-01
composition exerted its function through suppression of
galectin-1.
Example 2 In Vitro Suppression of Cancer Cells by the Combined
Treatment of TN-GL-01 Composition and an Anti-Cancer Drug
[0072] In this example, the effect of TN-GL-01 composition of
example 1.1 on the cell killing effect of a chemotherapeutic drug
(i.e., etoposide, doxorubicin, cisplatin, and paclitaxel) on
myeloma ARH-77 cells was investigated, in which cyclophosphamide
served as a comparative drug. Results are summarized in Tables 1 to
3, and FIGS. 3 and 4.
[0073] Reference is first made to Table 1, in which the data
indicated that TN-GL-01 composition was more effective than
cyclophosphamide in enhancing the etoposide induced cell death in
myeoloma ARH-77 cells (IC.sub.50 156 nM vs 238 nM, compared to the
control IC.sub.50 of 415 nM). Similar results are also observed for
another chemotherapeutic drug--doxorubicin (IC.sub.50 50 nM vs 73
nM, compared to the control IC.sub.50 of 99 nM)
TABLE-US-00001 TABLE 1 IC.sub.50 of myeoloma ARH-77 cells treated
with TN- GL-01 composition and a chemotherapeutic agent Treatment
IC.sub.50 (nM) Etoposide 415 Etoposide + Cyclophosphamide (3 mM)
238 Etoposide + TN-GL-01 (15 .mu.g/mL) 156 Doxorubicin 99
Doxorubicin + Cyclophosphamide (3 mM) 73 Doxorubicin + TN-GL-01 (15
.mu.g/mL) 50
[0074] To determine the effects of combined use of TN-GL-01 and any
other three drugs including paclitaxel, doxorubicin, and cisplatin,
the statistical differences between expected value and observed
value of the survival rate were calculated. If no significance
existed between the expected and observed values, the effect is
termed"additivity (p>0.05)". If the observed value of the
survival rate was significantly lower than the expected value, the
given effect in combination is termed "synergy (p<0.05)".
Additionally, antagonism indicates that a statistically higher
observed value of survival rate was found when comparing to its
expected value of survival rate. The expected value was determined
by multiplying the survival rate of each drug in specific dose. The
obtained value and value of each survival rate were measured by MMT
assay (FIG. 3 and FIG. 4). The results were presented as
mean.+-.standard deviation (SD) as shown in Tables 2 and 3.
[0075] Referring to FIG. 3, which depicts the respective effects of
TN-GL-01 composition, cisplatin, and doxorubicin on paclitaxel
induced cell death. Among the three anti-cancer agents (i.e.,
TN-GL-01 composition, cisplatin, and doxorubicin), TN-GL-01
composition was most effective in enhancing the paclitaxel induced
cell death (FIG. 3). Surprisingly, synergistic suppression of the
ARH-77 cells was observed when cells were treated with the
combination of TN-GL-01 composition (20 .mu.g/mL) and paclitaxel
(62.5 nM and 125 nM, respectively) (see Table 2).
[0076] Similarly, TN-GL-01 composition was also most effective in
enhancing the doxorubicin-induced cell death, as compared to that
of cisplatin or paclitaxel (FIG. 4). Also, synergistic suppression
of the ARH-77 cells was observed when cells were treated with the
combination of TN-GL-01 composition (20 .mu.g/mL) and doxorubicin
at the concentration up to 500 nM (Table 3), whereas in the case
when the concentration of doxorubicin was no more than 200 nM, then
only additive or antagonic effect was observed (Table 3).
TABLE-US-00002 TABLE 2 Cell Survival Rate Combination Expected
value observed value result Paclitaxel (62.5 nM) + 0.68 .times.
0.82 = 0.38 .+-. 0.02 Synergy TN-GL-01 (20 .mu.g/mL) 0.56 .+-. 0.03
(p = 0.002) Paclitaxel (125 nM) + 0.50 .times. 0.82 = 0.20 .+-.
0.03 Synergy TN-GL-01 (20 .mu.g/mL) 0.41 .+-. 0.03 (p = 0.0008)
Paclitaxel (62.5 nM) + 0.68 .times. 0.58 = 0.43 .+-. 0.03
Additivity Doxorubicin (100 nM) 0.39 .+-. 0.02 (p = 0.18)
Paclitaxel (125 nM) + 0.50 .times. 0.58 = 0.38 .+-. 0.01 Antagonism
Doxorubicin (100 nM) 0.29 .+-. 0.01 (p = 0.003) Paclitaxel (62.5
nM) + 0.68 .times. 0.65 = 0.48 .+-. 0.04 Additivity Cisplatin (3
.mu.M) 0.44 .+-. 0.04 (p = 0.27) Paclitaxel (125 nM) + 0.50 .times.
0.65 = 0.34 .+-. 0.01 Additivity Cisplatin (3 .mu.M) 0.33 .+-. 0.03
(p = 0.45)
TABLE-US-00003 TABLE 3 Cell Survival Rate combination Expected
value Observed value result Doxorubicin (250 nM) + 0.66 .times.
0.68 = 0.46 .+-. 0.02 Additivity TN-GL-01 (20 .mu.g/mL) 0.44 .+-.
0.02 (p = 0.46) Doxorubicin (500 nM) + 0.66 .times. 0.68 = 0.25
.+-. 0.01 Synergy TN-GL-01 (20 .mu.g/mL) 0.44 .+-. 0.03 (p =
0.0004) Doxorubicin (250 nM) + 0.66 .times. 0.65 = 0.56 .+-. 0.01
Antagonism Paclitaxel (40 .mu.M) 0.43 .+-. 0.02 (p = 0.0004)
Doxorubicin (500 nM) + 0.66 .times. 0.65 = 0.60 .+-. 0.01
Antagonism Paclitaxel (40 .mu.M) 0.43 .+-. 0.02 (p = 0.0003)
Doxorubicin (250 nM) + 0.66 .times. 0.69 = 0.53 .+-. 0.04
Antagonism Cisplatin (3 .mu.M) 0.45 .+-. 0.01 (p = 0.03)
Doxorubicin (500 nM) + 0.66 .times. 0.69 = 0.47 .+-. 0.03
Additivity Cisplatin (3 .mu.M) 0.45 .+-. 0.02 (p = 0.31)
[0077] Taken together, the results from this example suggested that
in addition to its own cytotoxicity on cancerous cells, the present
TN-GL-01 composition may result in synergistic suppression of
cancerous cells when administered together with another
chemotherapeutic agent, such as paclitaxel and doxorubicin.
Example 3 In Vivo Suppression of Ovarian Tumor by the Combined
Treatment of TN-GL-01 Composition and Paclitaxel
[0078] In this example, ovarian tumor was innoculated and inducted
in mice in accordance with procedures described in the "Material
and Methods" section. Mice were randomly assigned into different
groups and treated with the TN-GL-01 composition of example 1.1
(300 mg/Kg), paclitaxel (10 mg/Kg) or a combination of paclitaxel
(10 mg/Kg or 20 mg/Kg) and the TN-GL-01 composition (300 mg/Kg) via
intraperitoneal injection. Mice were then sacrificed, the
expression of galectin-1 in stromal cells, the amount of paclitaxel
retained in the cancerous tissues, as well as the level of CD-8
positive cells in the test animals were measured. Results are shown
in FIGS. 5 to 7.
[0079] By appearance, the growth of ovarian tumor excised from the
test animals treated with the TN-GL-01 composition of example 1.1
was significantly suppressed, as the weight of tumor was 52% less
than that of the control animals (FIGS. 5A and 5B). Further,
treatment with TN-GL-01 composition reduced the levels of
galectin-1 in stroma cells, which was consistent with the finding
in example 1.3. As to the expression of .alpha.-SMA, the number of
stromal cells positively expressed .alpha.-SMA was lower than that
of the control (FIG. 5A), which suggested that the TN-GL-01
composition might have changed the tumor microenvironment that
facilitated the infiltration of immunce cells, and was consistent
with the finding depicted in FIGS. 6A and 6B, in which an increased
number of immune cells (i.e., CD-8) was found in TN-GL-01
composition treated ovarian tumor.
[0080] It was also found that TN-GL-01 composition enhanced the
amount of a chemotherapeutic agent (i.e, paclitaxel) retained in
the tumor tissue. As depicted in FIG. 7A, the amount of paclitaxel
remained in the tumor tissue was significantly higher in tumors
treated with TN-GL-01 (300 mg/Kg) and paclitaxel (10 or 20 mg/Kg).
Accordingly, the tumor weight was also significantly reduced in
tumors treated with the combination of TN-GL-01 (300 mg/Kg) and
paclitaxel (10 or 20 mg/Kg) (FIG. 7B).
[0081] Taken together, results of the present disclosure indicate
that TN-GL-01 composition is suitable for the development of a
medicament or an adjuvant for treating cancers.
[0082] It will be understood that the above description of
embodiments is given by way of example only and that various
modifications may be made by those with ordinary skill in the art.
The above specification, examples and data provide a complete
description of the structure and use of exemplary embodiments of
the invention. Although various embodiments of the invention have
been described above with a certain degree of particularity, or
with reference to one or more individual embodiments, those with
ordinary skill in the art could make numerous alterations to the
disclosed embodiments without departing from the spirit or scope of
the present disclosure.
* * * * *